The object of the proposed research is to develop the use of immobilized enzymes which act in sequence so that they may be applied to the techniques of ultramicrobiochemical analyses through "enzymatic cycling." Enzymatic cycling, as presently carried out with soluble enzymes, makes it possible to measure pyridine nucleotides and some substances that are either oxidized or reduced by pyridine nucleotides, at levels as low as 10 to the minus 15th power M. By immobilizing the correct proportions of the two enzymes of the cycling reaction covalently to insoluble cellulose particles, it should be possible to greatly increase the effectiveness and use of enzymatic cycling for even more sensitive analyses. The immobilized enzyme preparation would be more stable and reusable. The analyses would be greatly simpler and shorter to carry out, and would provide more repeatable and reliable results. The primary effort will be focused around the technological and theoretical development of two enzymes acting in sequence while immobilized to the same particles. The application of this technique to biochemical analyses would allow for the potential analysis for even a few molecules of some substances--substances that are present in living systems or are synthesized in the laboratory in ranges too low to be detected by present technology.